Identification of combinatorial patterns of post-translational modifications on individual histones in the mouse brain.

1Medical Faculty, Brain Research Institute, University of Zürich and Department of Biology, ETH Zürich, Zürich, Switzerland.

Abstract

Post-translational modifications (PTMs) of proteins are biochemical processes required for cellular functions and signalling that occur in every sub-cellular compartment. Multiple protein PTMs exist, and are established by specific enzymes that can act in basal conditions and upon cellular activity. In the nucleus, histone proteins are subjected to numerous PTMs that together form a histone code that contributes to regulate transcriptional activity and gene expression. Despite their importance however, histone PTMs have remained poorly characterised in most tissues, in particular the brain where they are thought to be required for complex functions such as learning and memory formation. Here, we report the comprehensive identification of histone PTMs, of their combinatorial patterns, and of the rules that govern these patterns in the adult mouse brain. Based on liquid chromatography, electron transfer, and collision-induced dissociation mass spectrometry, we generated a dataset containing a total of 10,646 peptides from H1, H2A, H2B, H3, H4, and variants in the adult brain. 1475 of these peptides carried one or more PTMs, including 141 unique sites and a total of 58 novel sites not described before. We observed that these PTMs are not only classical modifications such as serine/threonine (Ser/Thr) phosphorylation, lysine (Lys) acetylation, and Lys/arginine (Arg) methylation, but also include several atypical modifications such as Ser/Thr acetylation, and Lys butyrylation, crotonylation, and propionylation. Using synthetic peptides, we validated the presence of these atypical novel PTMs in the mouse brain. The application of data-mining algorithms further revealed that histone PTMs occur in specific combinations with different ratios. Overall, the present data newly identify a specific histone code in the mouse brain and reveal its level of complexity, suggesting its potential relevance for higher-order brain functions.

A) Workflow for the isolation and analysis of long histone peptides from the mouse brain. B) Number of peptides identified for each histone subtype, in brackets the number of unique peptides identified and typical sequence coverage observed.

A) Table depicting each of the 304 combinatorial codes identified on H41–24 by ETD-MS. The number of each residue carrying a PTM is indicated at the top and each line represents an individual peptide. Probability of co-occurrence of (B) individual PTMs and (C) individual PTMs with groups of PTMs, on H41–23 determined by an association rule data-mining algorithm. The condition (left rows) is when a specific PTM is observed on H4, and the outcome (top columns) is the probability (indicated by a heat plot) that a second or several PTM(s) are observed at the same time on the same histone molecule.

Summary of N-terminal H2B and H2A combinatorial codes identified using ETD-MS. Each line represents an individual H2B1–25 (A) or H2A1–41 (C) peptide. Probability of co-occurrence of individual PTMs on H2B1–25 determined by an association rule data-mining algorithm (B). The condition (left rows) is when a specific PTM is observed on H2A/H2B, and the outcome (top columns) is the probability (indicated by a heat plot) that a second or several PTM(s) are observed at the same time on the same histone molecule. Diagram depicting the relationship between three PTMs on H2A1–41 (D). N-term Ac, K5ac and R3Me3 were either mutually exclusive or always seen in combination. The frequency of each PTM is indicated by the % above the circle, connections indicate the derived rule and its % occurrence. For instance, when K5ac was present, N-term ac was also observed in 100% of cases (connected by arrow), but R3me3 was never observed. When R3me3 was observed, N-term acetylation or K5ac was never observed (100% of cases), suggesting mutual exclusion of N-term acetylation and R3me3, potentially due to steric hindrance or conformational changes induced by each PTM.

A,B) Mass spectra of identified endogenous peptides with lysine propionylation and butyrylation and their synthetic counterparts.

Major peaks are labelled in the mass spectra and the fragment ions indicated in the peptide sequence using standard Mascot nomenclature . A) A novel site of lysine butyrylation on residue K95 of H2A. B) A novel site of lysine propionylation on residue K95 of H2A. C) Highly modified peptides that were detected using ETD-MS/MS included the N-terminal peptide from H4 ac-SGRGKacGGKacGLGKacGGAKacRHRKme2VLR, which contains 5 sites of acetylation and 1 site of dimethylation E) A novel site of lysine crotonylation on residue K108 of H2B.

Mass spectra of identified endogenous peptides with serine and threonine acetylation and their synthetic counterparts.

Major peaks are labelled in the mass spectra and the fragment ions indicated in the peptide sequence using standard Mascot nomenclature . A) A novel site of serine acetylation on residue S35 of H1. B) A novel site of threonine acetylation on residue T80 of H3. The reporter ion characteristic of the loss of an acetyl group (−80 Da) from the parent ion is indicated. C) A novel site of serine acetylation on S112 histone H1.

Summary of all novel PTMs identified on H1 (A), H2A (B), H2B (C), H3 (D) and H4 (E). Sites of PTMs are indicated by A for acetylation, B for butyrylation, Cr for crotonylation, Me1, Me2 and Me3 for mono-, di- and trimethylation, P for phosphorylation and Pr for propionylation. Residues are numbered starting with the first residue after the cleaved methionine. Canonical H1, H2A, H2B and H3 histones are shown which represent sequences common across all subtypes.